Hypoglycemia is the primary barrier to safe control of hyperglycemia in diabetes. Hypoglycemia can cause coma, seizures, accidents or even death. Glucagon counterregulation (GCR) is a key protection against hypoglycemia that is compromised in patients with type 1 diabetes through an unknown mechanism. We have previously confirmed that, in diabetic rats, pulsatile GCR can be amplified by intrapancreatic infusion of insulin and somatostatin if these signals are terminated during hypoglycemia [switch-off signals]. To explain these findings we proposed that the GCR response to hypoglycemia should be viewed as a response controlled by a pancreatic hormone network, rather than as an a-cell response to a specific signal and now we propose to study further this concept. To this end, we will identify additional signals that can restore GCR in -cell deficiency if they are infused in the pancreas and switched-off during hypoglycemia and determine the importance of the switch-off of the signal for GCR amplification. We will also establish the importance of these signals to GCR regulation in the normal pancreas. A major goal is to show that the complex mechanisms that control GCR can be explained by the following ensemble interactions: stimulation of -cells by increased blood glucose (BG) and inhibition of glucagon by elevated BG, -cell signals, and a-cell auto-feedback. We hypothesize that: [1] In STZ-treated rats in vivo, GCR can be amplified by withdrawal of several exogenous a- cell suppressing signals (switch-off); [2] In normal rats in vivo, glucagon-suppressing -cell signals play a key role in the control of GCR; [3] The proposed ensemble interactions explain most of GCR control, including the mechanisms of GCR dysregulation in -cell deficiency, and can be manipulated to repair the defective GCR in -cell deficiency. To verify these hypotheses we propose four Specific Aims. Aim 1 is to determine whether switch-off of multiple a-cell suppressing signals can augment defective GCR in insulinopenic rats; Aim 2 is to define the mechanism of GCR response to switch-off in -cell deficiency; Aim 3 is to establish the importance of different -cell signals and define the GCR control mechanism in normal rats; and Aim 4 is to determine how intrapancreatic or systemic a-cell suppressing signals modulate and whether they can repair the defective GCR response to hypoglycemia in -cell deficient rats. These clinically relevant studies are designed to understand the system-level control of GCR and suggest ways to enhance GCR in -cell-deficient diabetes. They bring novel concepts to the field by testing network control of GCR using a combination of experimental and modeling techniques. The results of the proposed efforts could provide strategies to repair GCR, which will use the subject's own glucagon stores. Ultimately, they may allow restoration of GCR in individuals with T1DM and others at high risk for severe hypoglycemia. PUBLIC HEALTH RELEVANCE: With a national and global diabetes epidemic, and increasing use of insulin as therapy, hypoglycemia will remain and may increase as a key barrier to safely achieving glycemic control for people with diabetes. The proposed studies will reveal new insights into the normal function and dysregulation of the complex endocrine mechanisms that prevent dangerous drops in blood glucose. This should allow future studies to develop novel therapeutic approaches to repair crucial defects in defenses against hypoglycemia for people with diabetes mellitus.